All living organisms require fundamental resources like carbohydrates, proteins, fats (macromolecules), water, and minerals for their growth and development.

This chapter primarily focuses on how plants obtain and utilize inorganic nutrients, the methods used to identify essential elements for plant growth, the criteria for determining essentiality, the specific roles of these elements, their deficiency symptoms, and their absorption mechanisms.

Additionally, the chapter provides a brief overview of the process and significance of biological nitrogen fixation.

Methods To Study The Mineral Requirements Of Plants

Historically, scientists studied how plants obtained nutrients from the soil. However, precise determination of which specific elements were essential became possible with the development of controlled culture methods.

In 1860, Julius von Sachs demonstrated for the first time that plants could successfully grow and reach maturity in a soil-free environment, using only a defined nutrient solution.

This technique of growing plants in a nutrient solution is known as hydroponics.

Hydroponics and similar methods are based on cultivating plants in a soil-free environment with precise control over the mineral composition supplied.

For these methods to be effective, highly purified water and mineral nutrient salts are essential. Impure water or salts could introduce unknown elements, making it difficult to determine the specific requirements of the plant being studied.

Through systematic experiments involving varying the composition or concentration of elements in the nutrient solution (adding, substituting, removing, or changing concentrations of specific elements), researchers were able to identify the elements essential for plant growth and development and to observe the symptoms that appear when a particular element is deficient.

Hydroponics has practical applications, such as the commercial production of vegetables like tomatoes, seedless cucumbers, and lettuce. For optimal growth in hydroponics, the nutrient solutions must be adequately aerated to provide oxygen to the roots; poor aeration would hinder root respiration and nutrient uptake.

Essential Mineral Elements

Plants absorb a wide variety of minerals from the soil. Analysis has shown that plants can contain over sixty of the 105 known elements. Some plants may even accumulate unusual elements like selenium or gold.

However, the mere presence of an element in a plant does not mean it is essential for that plant's growth or survival. Identifying which elements are truly necessary requires specific criteria.

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Criteria For Essentiality

An element is considered essential for plant growth and metabolism if it meets the following strict criteria:

1. The element is absolutely necessary for supporting normal growth and reproduction. Without this element, the plant cannot complete its life cycle (grow from seed to maturity, flower, and set seeds).
2. The requirement for the element must be specific. Its function cannot be completely replaced by supplying another element.
3. The element must be directly involved in the metabolic processes of the plant.

Based on these criteria, a select number of elements have been confirmed as essential for plants. These essential elements are further categorized based on the amount required by the plant tissues (in terms of dry matter):

1. Macronutrients: Required in large amounts (typically in excess of 10 mmole per Kg of dry matter).
   • Examples: Carbon (C), Hydrogen (H), Oxygen (O), Nitrogen (N), Phosphorus (P), Sulphur (S), Potassium (K), Calcium (Ca), and Magnesium (Mg).
   • Sources: C, H, and O are primarily obtained from CO$_2$ and H$_2$O. Others are absorbed from the soil as mineral ions.
2. Micronutrients (Trace elements): Required in very small amounts (typically less than 10 mmole per Kg of dry matter).
   • Examples: Iron (Fe), Manganese (Mn), Copper (Cu), Molybdenum (Mo), Zinc (Zn), Boron (B), Chlorine (Cl), and Nickel (Ni).

In addition to the 17 essential elements (9 macronutrients + 8 micronutrients), some elements are considered beneficial for higher plants, though not strictly essential according to the criteria. Examples include Sodium (Na), Silicon (Si), Cobalt (Co), and Selenium (Se).

Essential elements perform diverse functions and can be broadly grouped based on these roles:

1. Structural components: Elements that are components of biomolecules (like carbohydrates, proteins, nucleic acids) and hence are structural elements of cells (e.g., C, H, O, N).
2. Energy-related compounds: Elements that are part of energy-related chemical compounds (e.g., Magnesium in chlorophyll, Phosphorus in ATP).
3. Enzyme activators or inhibitors: Elements that function by activating or inhibiting specific enzymes.
   • Example activators: Mg$^{2+}$ activates Rubisco and PEP carboxylase (key enzymes in photosynthesis). Zn$^{2+}$ activates alcohol dehydrogenase. Mo activates nitrogenase (in nitrogen metabolism).
4. Osmotic potential alteration: Elements that affect the osmotic potential of a cell.
   • Example: Potassium (K$^+$) plays a crucial role in regulating the opening and closing of stomata by influencing turgor pressure. Minerals acting as solutes affect cell water potential.

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Role Of Macro- And Micro-Nutrients

Essential elements are involved in numerous metabolic processes, contributing to the overall function and health of the plant. Their roles include:

• Maintaining the permeability of the cell membrane.
• Regulating the osmotic concentration of cell sap.
• Functioning in electron-transport systems (like in photosynthesis and respiration).
• Acting as buffers to maintain pH stability.
• Serving as cofactors or activators in enzymatic activities.
• Being integral components of essential macromolecules and co-enzymes.

Specific functions of some essential elements:

• Nitrogen (N): Absorbed mainly as NO$_3^{-}$, also NO$_2^{-}$ or NH$_4^{+}$. Required in the greatest amount, especially by meristematic and metabolically active tissues. Major constituent of proteins, nucleic acids, vitamins, and hormones.
• Phosphorus (P): Absorbed as phosphate ions (H$_2$PO$_4^{-}$ or HPO$_4^{2-}$). Component of cell membranes (phospholipids), certain proteins, all nucleic acids, and nucleotides. Essential for phosphorylation reactions (e.g., in ATP).
• Potassium (K): Absorbed as K$^+$. Required abundantly in meristematic tissues, buds, leaves, and root tips. Helps maintain anion-cation balance, involved in protein synthesis, stomatal opening/closing, enzyme activation, and cell turgidity.
• Calcium (Ca): Absorbed as Ca$^{2+}$. Required by meristematic and differentiating tissues. Used in cell wall synthesis (as calcium pectate in middle lamella) and mitotic spindle formation. Accumulates in older leaves. Important for cell membrane function and regulation of metabolic activities.
• Magnesium (Mg): Absorbed as Mg$^{2+}$. Activates enzymes of respiration and photosynthesis. Involved in DNA and RNA synthesis. Constituent of the chlorophyll ring structure and helps maintain ribosome structure.
• Sulphur (S): Absorbed as sulphate (SO$_4^{2-}$). Present in amino acids cysteine and methionine. Constituent of coenzymes, vitamins (thiamine, biotin, Coenzyme A), and ferredoxin.
• Iron (Fe): Absorbed as ferric ions (Fe$^{3+}$). Required in larger amounts than other micronutrients. Important component of electron transfer proteins (ferredoxin, cytochromes). Involved in reversible oxidation (Fe$^{2+}$ to Fe$^{3+}$). Activates catalase enzyme. Essential for chlorophyll formation.
• Manganese (Mn): Absorbed as Mn$^{2+}$ ions. Activates enzymes in photosynthesis, respiration, and nitrogen metabolism. Essential for the splitting of water (photolysis) to produce oxygen during photosynthesis.
• Zinc (Zn): Absorbed as Zn$^{2+}$ ions. Activates various enzymes, especially carboxylases. Needed for auxin synthesis.
• Copper (Cu): Absorbed as cupric ions (Cu$^{2+}$). Essential for overall plant metabolism. Associated with enzymes in redox reactions, undergoes reversible oxidation (Cu$^+$ to Cu$^{2+}$).
• Boron (B): Absorbed as BO$_3^{3-}$ or B$_4$O$_7^{2-}$. Required for Ca$^{2+}$ uptake and utilization, membrane function, pollen germination, cell elongation, cell differentiation, and carbohydrate translocation.
• Molybdenum (Mo): Absorbed as molybdate ions (MoO$_4^{2-}$). Component of enzymes nitrogenase and nitrate reductase, crucial for nitrogen metabolism.
• Chlorine (Cl): Absorbed as chloride anion (Cl$^-$). Along with Na$^+$ and K$^+$, helps maintain anion-cation balance and solute concentration in cells. Essential for the water-splitting reaction in photosynthesis (oxygen evolution).

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Deficiency Symptoms Of Essential Elements

When the supply of an essential element falls below a certain level, plant growth is impaired. The concentration below which growth is reduced is called the critical concentration. An element is deficient when its concentration is below the critical concentration.

Lack of an essential element causes specific morphological changes in the plant, known as deficiency symptoms.

Symptoms vary depending on the element and disappear when the deficient element is supplied. Prolonged deficiency can lead to plant death.

Location of symptoms: The plant part showing symptoms depends on the element's mobility within the plant.

• Mobile elements: Deficiency symptoms appear first in older tissues. Elements like N, K, and Mg are actively moved from older, senescing leaves to younger, growing tissues.
• Immobile elements: Deficiency symptoms appear first in younger tissues. Elements like S and Ca are incorporated into structural components and are not easily transported out of mature tissues.

Common deficiency symptoms:

• Chlorosis: Loss of chlorophyll leading to yellowing of leaves. Caused by deficiency of N, K, Mg, S, Fe, Mn, Zn, Mo.
• Necrosis: Death of tissues, especially leaf tissue. Caused by deficiency of Ca, Mg, Cu, K.
• Stunted growth: Reduced overall plant size.
• Premature fall of leaves and buds.
• Inhibition of cell division: Caused by deficiency of N, K, S, Mo.
• Delayed flowering: Caused by low levels of N, S, Mo.

Identifying a specific deficiency can be complex as multiple symptoms can arise from one deficiency, and the same symptom can be caused by deficiency of different elements. Comparing observed symptoms across various plant parts with standard charts is necessary. Different plant species may also respond differently to the same deficiency.

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Toxicity Of Micronutrients

Micronutrients are required in very low amounts. While deficiency occurs when the concentration is too low, an increase beyond a certain moderate level can lead to toxicity.

There is a narrow range of concentration where micronutrients are optimal.

A mineral ion concentration is considered toxic if it reduces the dry weight of the plant tissue by about 10%. Critical toxicity concentrations vary among micronutrients and between different plant species.

Toxicity symptoms can be difficult to identify. Excess of one element can induce deficiency symptoms of another element by inhibiting its uptake or affecting its function.

Example: Manganese toxicity symptom is brown spots surrounded by chlorotic veins. Excess manganese can inhibit the uptake of iron and magnesium, compete with magnesium for enzyme binding, and inhibit calcium translocation to the shoot apex. Thus, manganese toxicity can actually cause iron, magnesium, and calcium deficiencies.

Mechanism Of Absorption Of Elements

Studies on element absorption by plants (using isolated cells, tissues, or organs) show that the process involves two main phases:

1. Initial phase (Passive uptake): Rapid uptake of ions into the 'free space' or apoplast (cell walls and intercellular spaces) of root cells. This movement is passive, often occurring through ion channels (selective transmembrane protein pores).
2. Second phase (Active uptake): Slower uptake of ions into the 'inner space' or symplast (cytoplasm and vacuole) of root cells. This movement requires the expenditure of metabolic energy (ATP) and is an active process mediated by membrane transport proteins (pumps).

The movement of ions is called flux. Inward movement into cells is influx, and outward movement is efflux.

Both passive and active transport mechanisms are involved in the uptake of mineral nutrients from the soil by root cells.

Translocation Of Solutes

After mineral salts are absorbed by the roots and enter the xylem, they are transported upwards to the rest of the plant. This translocation occurs through the xylem along with the ascending stream of water, which is pulled up by the transpirational pull (Chapter 11).

Analysis of xylem sap confirms the presence of mineral salts. Use of radioisotopes has also shown that minerals are transported via the xylem.

The distribution of minerals to different plant parts is determined by areas of high demand (sinks), primarily the growing regions (meristems, young leaves, developing reproductive structures, storage organs).

Unloading of minerals at sinks occurs at the fine vein endings, through diffusion or active uptake by the sink cells.

Remobilisation of minerals from older tissues to younger, growing tissues is common for mobile elements (N, K, P, S). Immobile elements (like Ca) are not remobilised.

While traditionally thought that xylem only transports inorganic nutrients and phloem only organic ones, xylem sap analysis shows that some nitrogen and small amounts of P and S are transported in organic forms (amino acids, amides). There is also some exchange of materials between xylem and phloem, blurring this strict distinction.

Soil As Reservoir Of Essential Elements

Most essential nutrients for plants originate from the weathering and breakdown of rocks in the Earth's crust. These geological processes release dissolved ions and inorganic salts that enrich the soil.

The study of the role of elements derived from rock minerals in plant nutrition is called mineral nutrition.

Soil is a complex medium that provides much more than just mineral nutrients. It also:

• Harbours nitrogen-fixing bacteria and other beneficial microbes.
• Holds water essential for plant life.
• Provides air (oxygen) to the roots for respiration.
• Acts as a stable matrix that anchors and supports the plant.

Because the availability of essential minerals in the soil directly affects crop yield, fertilizers containing both macronutrients (N, P, K, S, etc.) and micronutrients (Cu, Zn, Fe, Mn, etc.) are often applied to supplement the soil's mineral content.

Metabolism Of Nitrogen

Nitrogen is one of the most crucial elements for life, second only to carbon, hydrogen, and oxygen in abundance in living organisms. It is a vital component of amino acids, proteins, nucleic acids, hormones, chlorophyll, and many vitamins.

Nitrogen is often a limiting nutrient for plant growth in both natural and agricultural ecosystems, as its availability in the soil is limited, and plants must compete for it with microbes.

Atmospheric nitrogen exists as N$_2$ molecules, held together by a very strong triple covalent bond (N $\equiv$ N).

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Nitrogen Cycle

The nitrogen cycle describes the complex series of processes that convert nitrogen between different forms in the environment (atmosphere, soil, biomass) (Figure 12.3).

Key processes in the nitrogen cycle:

1. Nitrogen Fixation: The conversion of atmospheric nitrogen gas (N$_2$) into ammonia (NH$_3$).
   • Atmospheric fixation: Non-biological processes like lightning and UV radiation convert N$_2$ to nitrogen oxides. Industrial combustion, forest fires, vehicle exhausts also produce nitrogen oxides.
   • Biological nitrogen fixation: Conversion of N$_2$ to ammonia by living organisms (certain prokaryotes).
2. Ammonification: The decomposition of organic nitrogenous matter (from dead plants and animals) into ammonia by microorganisms. Some ammonia evaporates, but most is converted further in the soil.
3. Nitrification: The process where ammonia is converted into nitrate (NO$_3^{-}$) in the soil through the activity of specific nitrifying bacteria.
   • Step 1: Ammonia is oxidized to nitrite (NO$_2^{-}$) by bacteria like *Nitrosomonas* and *Nitrococcus*. ($\textsf{2NH}_3 + \textsf{3O}_2 \rightarrow \textsf{2NO}_2^{-} + \textsf{2H}_2\textsf{O} + \textsf{2H}^+$)
   • Step 2: Nitrite is further oxidized to nitrate (NO$_3^{-}$) by bacteria like *Nitrobacter*. ($\textsf{2NO}_2^{-} + \textsf{O}_2 \rightarrow \textsf{2NO}_3^{-}$)

   These nitrifying bacteria are chemoautotrophs (obtain energy from chemical reactions).

4. Assimilation: Plants absorb nitrate (the primary form, also some nitrite and ammonium) from the soil. Nitrate is then transported to leaves and reduced back to ammonia before being incorporated into amino acids and other organic nitrogen compounds.
5. Denitrification: The reduction of nitrate present in the soil back into nitrogen gas (N$_2$) by certain bacteria, such as *Pseudomonas* and *Thiobacillus*. This process returns nitrogen to the atmosphere.

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Biological Nitrogen Fixation

Only certain prokaryotic organisms (bacteria, cyanobacteria) possess the enzyme nitrogenase, which catalyzes the reduction of atmospheric nitrogen (N$_2$) to ammonia (NH$_3$). Organisms capable of this are called N$_2$-fixers.

The reaction catalyzed by nitrogenase is: N $\equiv$ N + 8e$^-$ + 8H$^+$ + 16 ATP $\rightarrow$ 2NH$_3$ + H$_2$ + 16 ADP + 16 Pi

Nitrogen-fixing microbes can be:

• Free-living: Living independently in soil or water.
  • Aerobic: *Azotobacter*, *Beijerinckia*.
  • Anaerobic: *Rhodospirillum*.
  • Cyanobacteria (some free-living, also photosynthetic): *Anabaena*, *Nostoc*.
• Symbiotic: Forming mutually beneficial associations with plants. The most prominent is the legume-bacteria association.

Symbiotic Nitrogen Fixation (Legume-Bacteria Relationship):

The bacterium Rhizobium forms symbiotic associations with the roots of leguminous plants (e.g., peas, beans, clover), resulting in the formation of root nodules (small outgrowths on the roots).

Other microbes, like *Frankia*, form similar nitrogen-fixing nodules on the roots of non-leguminous plants (e.g., *Alnus*).

Both Rhizobium and Frankia are free-living in soil but fix nitrogen only when in symbiotic association with host plants.

Nodules: Cutting open a legume root nodule reveals a red or pink color due to the presence of leg-haemoglobin. This pigment is an oxygen scavenger that protects the nitrogenase enzyme from molecular oxygen.

Nodule Formation Process (Figure 12.4):

1. Rhizobia bacteria multiply near the root and attach to root hair cells.
2. Root hairs curl in response to bacterial signals, and bacteria invade the curled root hair.
3. An infection thread is formed, carrying bacteria into the root cortex.
4. Bacteria are released into cortical cells and differentiate into specialized nitrogen-fixing cells called bacteroids.
5. Division and growth of inner cortical and pericycle cells are stimulated, leading to the formation of the nodule.
6. The mature nodule develops a direct vascular connection with the host root for nutrient exchange.

The nodule provides an environment for nitrogen fixation. It contains the nitrogenase enzyme complex and leg-haemoglobin.

Nitrogenase enzyme: A Mo-Fe protein. It is highly sensitive to oxygen and requires anaerobic conditions for activity. Leg-haemoglobin acts as an oxygen scavenger within the nodule, binding free oxygen and maintaining the low oxygen concentration necessary for nitrogenase to function.

Energy for nitrogen fixation: The reduction of N$_2$ to ammonia requires a high input of ATP energy (16 ATP per 2 NH$_3$). This energy is provided by the respiration of the host plant cells within the nodule.

Fate of ammonia: At physiological pH, ammonia (NH$_3$) is immediately converted to ammonium ion (NH$_4^+$). While plants can absorb ammonium, it is toxic at high concentrations. NH$_4^+$ is rapidly assimilated into organic compounds, primarily amino acids, within the plant.

Assimilation of ammonium into amino acids occurs mainly through two ways:

1. Reductive amination: Ammonium ion reacts with $\alpha$-ketoglutaric acid (a keto acid) to form glutamic acid. The enzyme involved is glutamate dehydrogenase. ($\alpha$-ketoglutaric acid + NH$_4^+$ + NADPH $\rightarrow$ Glutamic acid + H$_2$O + NADP).
2. Transamination: The amino group of one amino acid (often glutamic acid) is transferred to the keto group of another keto acid to form a new amino acid. This reaction is catalyzed by enzymes called transaminases. Glutamic acid plays a central role as the main amino acid from which amino groups are transferred. (Glutamic acid + Keto acid $\rightarrow$ another amino acid + $\alpha$-ketoglutaric acid).

Amides (Asparagine, Glutamine): Formed from aspartic acid and glutamic acid, respectively, by adding another amino group. Amides contain more nitrogen than amino acids and are important forms for transporting nitrogen within the plant via xylem vessels. Nodules of some plants (like soybean) export fixed nitrogen as ureides, which also have a high nitrogen to carbon ratio.

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